Facile laboratory method for localising biomolecules to the surface of cells and viruses

ABSTRACT

A facile method of localising sulfhydryl (—SH) group containing biomolecules, in particular peptides, to the membranes of living cells and enveloped viruses and a kit for use in a biological laboratory in accordance with the method.

FIELD OF INVENTION

The invention relates to a facile method of localising sulfhydryl (—SH)group containing biomolecules, in particular peptides, to the membranesof living cells and enveloped viruses and a kit for use in a biologicallaboratory in accordance with the method.

BACKGROUND ART

Historically, the capabilities of biological and chemical laboratorieshave resulted in biological and chemical research being performed atseparate locations. Bioconjugation is the process of coupling twobiomolecules together in a covalent linkage (Hermanson (2008)).Bioconjugation is employed to study the function of biomolecules inliving systems. The methods employed require the capabilities of bothbiological and chemical laboratories.

Water dispersible constructs of the general structure F-S-L (where F isa functional moiety, S is a spacer and L is a lipid) that spontaneouslyincorporate into lipid bilayers (KODE™ Constructs) have providedresearchers with a means of functionalising the surface of living cellsand membrane bound organelles and virions without the need for thecapabilities of a chemical laboratory (Bovin et al (2005), Carter et al(2007), Korchagina et al (2008) and Bovin et al (2009)).

Notwithstanding these advances, the use of KODE™ Constructs byresearchers in biological laboratories is for the most part restrictedto the use of KODE™ Constructs that are available commercially. A methodof preparing bespoke KODE™ Constructs capable of being employed in abiological laboratory using the equipment routinely available in thosefacilities is desirable.

Is an object of the invention described in this specification to providesuch a method. Is an object of the invention described in thisspecification to provide a kit to facilitate the use of such method.These objects are to be read disjunctively with the object to at leastprovide a useful choice.

STATEMENT OF INVENTION

In a first aspect the invention provides a method of localising asulfhydryl (—SH) group containing biomolecule to a surface comprisingthe steps:

-   -   1. Mixing in a volatile reaction buffer a molar excess of the        sulfhydryl (—SH) group containing biomolecule with a lipid        conjugated maleimide of the structure F-S-L to provide a        reaction mix;    -   2. Incubating the reaction mix for a time and at a temperature        sufficient to allow substantially all the lipid conjugated        maleimide to have reacted;    -   3. Drying the reaction mix to remove the reaction buffer and        provide a reaction product; and    -   4. Contacting a solution of the reaction product with the        surface,        where:    -   F is

-   -   S is selected from the group consisting of:

-   -   L is an amide linked diacyl- or dialkyl-glycerophospholipid,    -   m is the integer 1, 2 or 3,    -   M is a monovalent cation or substituent,    -   n is the integer 1, 2 or 3,    -   p is the integer 1, 2 or 3,    -   q is the integer 3, 4 or 5, and    -   the median value of r is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or        16.

Preferably, the sulfhydryl (—SH) group is a cysteine residue (Cys).

Preferably, the sulfhydryl (—SH) group containing biomolecule is apeptide. More preferably, the sulfhydryl (—SH) group containingbiomolecule is a peptide with a cysteine residue (Cys) at or proximal tothe C-terminus of the peptide. Most preferably, the sulfhydryl (—SH)group containing biomolecule is a peptide with a cysteine residue (Cys)at the C-terminus.

Preferably, the surface is a membrane. More preferably, the surface is acell membrane or the membrane of an enveloped virus. Yet morepreferably, the surface is a cell membrane. Most preferably, the surfaceis the cell membrane of a red blood cell.

The method permits the localising of sulfhydryl (—SH) group containingbiomolecules to the membranes of living cells.

Preferably, the incubating is for a time greater than 8 hours and atroom temperature (circa 25° C.).

Preferably, the drying is by freeze-drying.

Preferably, the solution of the reaction product is an aqueous solutionof the reaction product. More preferably, the solution of the reactionproduct is an aqueous saline solution of the reaction product.

The method excludes a step of purifying the reaction product byconventional separation methods such as column chromatography prior topreparing the solution of the reaction product and the step ofcontacting the solution of the reaction product with the surface.

Preferably, the volatile reaction buffer is 0.1M 4-methylmorpholineformate (4-MMF) in 30% isopropanol (IPA) at a pH in the range 6.5 to6.7.

Preferably, m is the integer 2. Preferably, n is the integer 2.Preferably, p is the integer 2. Preferably, q is the integer 4. Morepreferably, each of m, n and p are the integer 2 and q is the integer 4.Preferably, the median value of r is 6.

Preferably, L is an amide linked diacylglycerophospholipid. Morepreferably, L is an amide linked phosphatidylethanolamine or aphosphatidylserine. Yet more preferably, L is an amide linkedphosphatidylethanolamine. Most preferably, L is1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE).

Preferably,

-   -   S is

Preferably, M is H or CH₃. More preferably, M is H.

In a second aspect the invention provides a kit packaged with directionsfor use in a method of localising a sulfhydryl (—SH) group containingbiomolecule to a surface comprising:

-   -   1. an amount of a lipid conjugated maleimide of the structure        F-S-L; and    -   2. a volume of a volatile reaction buffer,        where:    -   F is

[followed by page 5]

-   -   S is selected from the group consisting of:

-   -   -   and

-   -   L is an amide linked diacyl- or dialkyl-glycerophospholipid,    -   m is the integer 1, 2 or 3,    -   M is a monovalent cation or substituent,    -   n is the integer 1, 2 or 3,    -   p is the integer 1, 2 or 3,    -   q is the integer 3, 4 or 5, and    -   the median value of r is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or        16.

Preferably, the sulfhydryl (—SH) group is a cysteine residue (Cys).

Preferably, the sulfhydryl (—SH) group containing biomolecule is apeptide. More preferably, the sulfhydryl (—SH) group containingbiomolecule is a peptide with a cysteine residue (Cys) at or proximal tothe C-terminus of the peptide. Most preferably, the sulfhydryl (—SH)group containing biomolecule is a peptide with a cysteine residue (Cys)at the C-terminus.

Preferably, the volatile reaction buffer is 0.1M 4-methylmorpholineformate (4-MMF) in 30% isopropanol (IPA) at a pH in the range 6.5 to6.7.

Preferably, m is the integer 2. Preferably, n is the integer 2.Preferably, p is the integer 2. Preferably, q is the integer 4. Morepreferably, each of m, n and p are the integer 0.2 and q is the integer4. Preferably, the median value of r is 6.

Preferably, L is an amide linked diacylglycerophospholipid. Morepreferably, L is an amide linked phosphatidylethanolamine or aphosphatidylserine. Yet more preferably, L is an amide linkedphosphatidylethanolamine. Most preferably, L is1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE).

Preferably,

-   -   S is

Preferably, M is H or CH₃. More preferably, M is H.

In the description and claims of this specification the followingacronyms, terms and phrases have the meaning provided:

“Comprising” means “including”, “containing” or “characterized by” anddoes not exclude any additional element, ingredient or step.

“Consisting of” means excluding any element, ingredient or step notspecified except for impurities and other incidentals.

“Localised” means associated with a surface by non-covalent interactionsbetween the molecule comprising the localised entity and the surface(and “localising” and “localisation” have a corresponding meaning).

“Kodecyte” means a cell modified by incorporation into the cell membraneof a construct of the general structure F-S-L (where F is a functionalmoiety, S is a spacer selected to provide a water dispersible constructand L is a lipid).

“Kodevirion” means an enveloped virus particle modified by incorporationinto the enveloping membrane of a construct of the general structureF-S-L (where F is a functional moiety, S is a spacer selected to providea water dispersible construct and L is a lipid).

“PBS” denotes phosphate buffered saline.

“Proximal” means in the vicinity of the specified location. For theavoidance of doubt the phrase “proximal to the C-terminus of thepeptide” means within x amino acid residues of the C-terminus (where thepeptide consists of ‘x’ amino acid residues).

“Reaction product” means the product of a reaction prior topurification.

“Saline” means a solution of one or more salts.

“Synthetic” means prepared by chemical synthesis.

“Water soluble” means, in the context of describing the properties ofconstructs of the general structure F-S-L (where F is a functionalmoiety, S is a spacer and L is a lipid), a stable, single phase systemis formed when the construct is contacted with water or saline (such asPBS) at a concentration of at least 100 μg/mL at a temperature of 25° C.in the absence of organic solvents or detergents. The terms “soluble”and “dispersible” are used synonymously.

It will be recognised that the phosphate moiety of a phospholipid isoften protonated, but the proton (H⁺) may be replaced by anothermonovalent cation such as Na⁺, K⁺, NH₄ ⁺ or triethylamine([NH(CH₂CH₃)₃]⁺). Similarly, the primary and secondary amino functionsof a construct may also be protonated. The constructs prepared accordingto the method described in this specification may therefore be preparedas a range of salts, including pharmaceutically acceptable salts.

The carboxylate residues of the moiety designated ‘CMG’ (Bovin et al(2009)) may be protonated, associated with another monovalent cationsuch as Na⁺, K⁺, NH₄ ⁺ or triethylamine ([NH(CH₂CH₃)₃]⁺) or covalentlybound by amidation or esterification to a monovalent substituent toprovide a substituted derivative such as the moiety designated ‘MCMG’.

where the suffix “-al” is employed in respect of the substituents R₁ andR₂ of a diacyl lipid, an aldehyde structure is intended as exemplifiedby cis-9-octadecenal of the structure:

Where the suffix “-yl” is employed in respect of the substituents R₁ andR₂ of a dialkyl lipid, an alkyl structure is intended as exemplified bycis-9-octadecenyl of the structure:

In the absence of the stereochemistry at a chiral centre being shown,the representations of the structures of compounds comprising one ormore chiral centres encompass the distereoisomers, enantiomers andmixtures thereof of the compounds, with the proviso that where therepresentation is of the structure of a compound or a portion of thestructure of a compound of biological origin, the structure or portionof the structure represented is limited to structures or portions ofstructures that are functionally equivalent to the structure or portionof the structure of the compound of biological origin.

An asterisk (*) is used to represent a point of covalent attachment toanother moiety and does not indicate an atom. Specifically, in therepresentations of the moieties F, S and L of the lipid conjugatedmaleimide of the structure F-S-L the asterisk denotes the point ofcovalent attachment to the immediately adjacent moiety.

The amino acid residues of peptides are identified according to Table 3of Appendix 2 of Annex C of the Administrative Instructions under thePatent Cooperation Treaty dated 7 Feb. 2007 and in accordance with theconvention:

-   -   [“amino (N) terminus”]H₂N-XaaXaaXaa . . . XaaXaaXaa-COOH        [“carboxy (C) terminus”]

The terms “first”, “second”, “third”, etc. used with reference toelements, features or integers of the subject matter defined in theStatement of Invention and Claims, or when used with reference toalternative embodiments of the invention are not intended to imply anorder of preference.

Where concentrations or ratios of reagents are specified theconcentration or ratio specified is the initial concentration or ratioof the reagents. Where values are expressed to one or more decimalplaces standard rounding applies. For example, 1.7 encompasses the range1.650 recurring to 7.499 recurring.

The invention will now be described with reference to a General Example,a Specific Example, and the figures of the accompanying drawings pages.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Structure of the lipid conjugated maleimide designatedFSL-RFG(Mal).

FIG. 2. Schematic overview of a method according to that described inthe General Example.

FIG. 3. Serology of kodecytes prepared as described in the SpecificExample using SYPH positive sera E47 (3A and 3B) and SYPH negative seraE216 (3C and 3D). Abbreviations on gel cards correspond to kodecytesprepared using the construct designated FSL-SYPH3 (Henry et al (2010))(‘FS’) and the construct designated FSL-RFG(Mal)-SYPH3 (‘RFG’).

FIG. 4. Thin-layer chromatography of the peptide designated SYPH3 andsamples from batches of the constructs designated FSL-SYPH3 (Henry et al(2010)), FSL-RFG(Mal) and FSL-RFG(Mal)-SYPH3: FSL-SYPH3 (Batch #1) [lane1]; FSL-RFG(Mal)SYPH3 (Batch #1)[lane #2]; FSL-RFG(Mal)SYPH3 (Batch#2)[lane #3]; FSL-RFG(Mal) [lane #4]; SYPH3 peptide (diluted in 4MMF)[lane #5] and SYPH3 peptide (diluted in water) [lane #6].

DETAILED DESCRIPTION

Weinberg et al (2009) and Bovin et al (2009) both describe thepreparation of peptide-lipid constructs for use in the preparation ofkodecytes to be used in assays for the presence of reactive antibody.Both publications describe the preparation of peptide-lipid constructsvia a lipid conjugated maleimide intermediate. Weinberg et al (2009)describes the preparation and use as the intermediate of amaleimido-derivative of DOPE-PEG₆-NH₂. Bovin et al (2009) describes thepreparation and use as the intermediate of a construct designatedFSL-RFG(Mal) (FIG. 1). This latter intermediate is prepared by thetreatment of the construct designated DOPE-Ad-CMG(2)amine with a 5-foldexcess of 3-maleimidopropionic acid oxybenztriazol ester inisopropanol-water.

Consistent with good laboratory practice the peptide-lipid constructsprepared by the methods disclosed in these publications are purifiedprior to their use in the preparation of the kodecytes required for usein diagnostic or prognostic assays. However, the step of purification byseparation methods such as chromatography invariably results in lossesof construct which is undesirable, particularly where the peptide islimited in amount, or the kodecytes to be prepared are only forexperimental purposes.

In the method of the present invention the requirement for purificationof the construct prior to use in the preparation of the kodecytes may becircumvented in part by the use of a reaction buffer that is removableby volatilization. The removal of the reaction buffer can beconveniently achieved in a biological laboratory, particularly if theprimary focus of that laboratory is cell biology, by freeze-drying(lyophilisation).

Following incubation of the reaction mix and drying according to themethod of the invention, the incorporation of the peptide-lipidconstruct into the membrane of the cell (kodecyte) or enveloped virus(kodevirion) serves as the separation step that has otherwise beenachieved by the chromatography. The method of the invention circumventsthe need for this intermediate purification step.

The method of the invention also permits the provision of a kit that maybe conveniently employed in laboratories lacking equipment or experiencein the purification of synthetic reaction products.

General Example Step 1—Preparation of Reaction Mix

In accordance with the method of the invention a quantity of sulfhydryl(—SH) group containing peptide, e.g. a Cys containing peptide,sufficient to provide at least a 20% molar excess relative to thequantity of lipid conjugated maleimide to be used is prepared.Typically, synthetic peptides are supplied with quoted purities of >95%and a 20% excess should be sufficient for the reaction with the lipidconjugated maleimide to be complete. However, it will be recognised thatquality and purity of peptides may vary between suppliers attributableto factors including cysteine (Cys) residue oxidation and small volumeweighing errors. For these reasons users of the method of the inventionmay wish to employ up to a 100% molar excess of peptide relative to thequantity of lipid conjugated maleimide to be used to ensure nearquantitative yield of peptide-lipid construct.

If necessary, spectrophotometric determination of reactive sulfhydrylcontent (C_(SH)) (μmol HS/mg) may be determined according to knownprocedures (Anon (2011)). If the determined C_(SH) is too low thereduction of disulfide may be considered (Anon (2007)). The use oftributyl phosphine or TCEP should be avoided due to the reportedreactivity with maleimide (Shafer et al (2000); Tyagarajan et al(2003)). If spectrophotometric determination of C_(SH) is not performedusers of the method of the invention are recommended to assume a purityof the synthetic peptide of no greater than 75% for the purpose ofstoichiometric calculations (Cook (2006)).

It will be recognised from the foregoing that immunoglobulins arecandidate sulfhydryl containing peptides that may potentially be used inthe method of the invention. The indigenous disulfide groups in thehinge region of the immunoglobulin structure may be reduced with lowconcentrations of DTT, TCEP or MEA while leaving the disulfide bondsbetween the heavy and light chains relatively intact (Sun et al (2005)).As noted in Hermanson (2008) similar reduction can be done with F(ab′₂)fragments produced from the pepsin digestion of immunoglobulin G (IgG)molecules.

As noted elsewhere in this specification the phrase “sulfhydryl (—SH)group containing biomolecules” will be understood to encompassbiomolecules where the requisite sulfhydryl group is natural, generated(i.e. by reduction of an existing disulfide) or introduced. Methods forthe introduction of sulfhydryl groups in biomolecules using reagentssuch as N-acetyl, homocysteine thiolactone, AMBH, cystamine, SAMSA,SATA, SATP, SMPT and SPDP are known (Hermanson (2008)).

Irrespective of its source the quantity of sulfhydryl containing peptideis added to a volume of volatile reaction buffer (0.1 M4-methylmorpholine formate (4-MMF) in 30% isopropanol (IPA) at a pH inthe range 6.5 to 6.7) from a freshly opened, sealed vial and sonicateduntil a clear solution is obtained. An initial volume of 300 μl of thevolatile reaction buffer is recommended for a quantity of peptidecorresponding to 1 μmol (100% molar excess with respect to the lipidconjugated maleimide). The volatile reaction buffer should be storeddegassed in a nitrogen atmosphere.

If necessary, but with caution, a further volume of buffer may be addedor mixtures (1:1; v/v) of trifluoroethanol-volatile reaction buffer,hexafluroisopropanol-volatile reaction buffer, 2-methoxyethanol-volatilereaction buffer, trifluoroethanol-pyridine,hexafluoroisopropanol-pyridine, 2-methoxyethanol-pyridine, neat DMF or 6guanidinium hydrochlorides may be used for dissolving peptides thatotherwise prove insoluble at sufficiently high concentration.

Checking the extent of peptide solubility in the volatile reactionbuffer before proceeding with the reaction is recommended. Note thatDMSO should not be used to promote dissolution of the peptide as it maypromote disulfide bond formation. Complete dissolution of the peptide isa prerequisite for successful reaction with the lipid conjugatedmaleimide.

An amount of the lipid conjugated maleimide (0.5 μmol) is reconstitutedin a volume (100 μl) of the volatile reaction buffer from a freshlyopened vial and following mixing bysonicating or vortexing for oneminute a volume (300 μl) of the solution of peptide prepared as aboveadded to the volume of lipid conjugated maleimide.

Step 2—Incubation the Reaction Mix

The reaction mix is sonicated in the sealed vial for 10 to 60 minutes inan ultrasonic bath. Typically, a clear solution will be obtained, butsome partial precipitation may occur to provide a cloudy solution orfine suspension, particularly if the peptide being used is a basicpeptide. In such circumstances periodic sonication over a period up totwo hours should be sufficient to achieve adequate dissolution.

The mixture is incubated away from direct light for a time andtemperature sufficient to allow all the lipid conjugated maleimide toreact, e.g. overnight at at room temperature (circa 25° C.).

Step 3—Drying the Reaction Mix

Optionally, the incubation is followed by the quenching of any possiblyunreacted lipid conjugated maleimide by the addition of anon-interfering sulfhydryl containing compound such as cysteine (Cys),DTT, ME, etc. prior to freeze-drying. For example, the addition of avolume (60 μl) of 0.1 M mercaptoethanol may be added with stirring forat least one hour prior to the addition of 1 mL of water, freezing anddrying.

Note that a vacuum centrifuge is not to be used. In addition, drying thereaction product using Fisher apparatus at elevated temperatures, orwashing with absolute ether can yield a dried reaction product that isdifficult to reconstitute in water.

For aliquoting and storage the dried reaction product is reconstitutedin water by the addition of one drop of ethanol and a volume (1 mL) ofwater. Aliquots may then be freeze-dried as before in ready-to-usequantities. Immediate freeze-drying is recommended in the event theproduct is unstable in water or subject to degradation by abiotic orbiotic factors. The reaction product following freeze-drying should bean amorphous white powder that is ready to use in the preparation ofkodecytes.

Step 4—Contacting a Solution of the Reaction Product with a Surface

As noted above the method of the invention circumvents the need topurify the reaction product by separation methods (e.g. chromatography)and is therefore particularly convenient for use in a researchlaboratory with limited equipment, or equipment that is not suited toanalytical and semipreparative scale purification. The reaction productis virtually free of by-products and contaminants that will interferewith the preparation of the kodecytes.

The preparation of kodecytes exploits the properties of syntheticconstructs of the general structure F-S-L that are readily dispersiblein biocompatible media; including saline, yet spontaneously incorporateinto cell membranes. In effect, the purification is achieved in themethod of the present invention by the incorporation of the construct inthe cell membrane, any by-products and contaminants remaining dissolvedin the suspending medium, e.g. a cell preservative (CELPRESOL™).

Kodecytes may be readily separated from the medium and washed withoutloss of construct as described elsewhere (Weinberg et al (2009); Bovinet al (2009)).

Presented with the Specific Example one could readily establish whatvariations to the exemplifying volatile reaction buffer (0.1 M4-methylmorpholine formate (4-MMF) in 30% isopropanol (IPA) at a pH inthe range 6.5 to 6.7) provided comparable results.

It is anticipated that volatile reaction buffers other than thatspecified above may be used. In addition to moderate variations in theconcentrations of 4-MMF and IPA, these components of the reaction buffermay be substituted by other components. The amine (cationic) component(4-MMF) could be replaced by 4-ethylmorpholine, triethylamine,trimethylamine, N-methylpiperidine or N-methylpyrrolidine and theorganic co-solvent (IPA), although preferred, could be replaced, atleast in part, by n-propanol, ethanol, methanol, t-butanol,2-methoxyethanol or trifluoroethanol. Where other co-solvents areincluded in the volatile reaction buffer the total concentration ofco-solvent should be in the range 20 to 60%.

It is also anticipated that In addition to variations in theconcentration of the components 4-MMF and IPA (and their substitution byother components) other buffering systems may be employed. However,trifluoroacetic acid (TFA) and hydrochloric acid (HCl) should not beused as components of these other buffering systems. These components(TFA and HCl) yield salts that are non-volatile at room temperature andare therefore best avoided. The molarity of the buffering system shouldbe in the range 0.05 to 0.3 M and the pH in the range 5.8 to 7.5 whenother buffering systems are employed.

Specific Example

The properties of a peptide-lipid construct and kodecytes preparedtherefrom according to the method of the invention were compared withthe properties of the same peptide-lipid construct (FSL-SYPH3) andkodecytes prepared therefrom according to known methods (Henry et al(2010)).

Materials

Blood group O packed red blood cells (RBCs) were washed with phosphatebuffered saline (PBS). Washed RBCs were suspended in cell preservative(CELPRESOL™). PBS for the preparation of dilutions of constructs wasfilter sterilised. Stock solutions were prepared at a concentration of200 μg/mL in PBS. Positive (POS) and negative (NEG) sera were chosenfrom a panel that had been previously determined by RPR, TPHA, and TPPA.

Methods and Results

A volume of 2 mL of filter sterilised PBS was added to a vial containing400 μg of the construct designated FSL-SYPH3 to provide a stock solutionof 200 μg/mL. The dispersion was vortexed and warmed to 37° C. for tenminutes. A stock solution of the construct designated FSL-RFG(Mal)-SYPH3 was similarly prepared at the same concentration.

Dilution series for each of the constructs were prepared atconcentrations of 25, 50, 100 and 200 μg/mL. Equal volumes (30 μL) ofthe packed group O RBCs were added to and mixed with each of thedilutions of the dilution series for each construct in glass tubes. Thetubes were then incubated in a water bath at 37° C. for 120 minutes withintermittent gentle mixing (three times) during the incubation period.The incubated RBCs were then washed two times with PBS and one time withcell preservative (CELPRESOL™).

A cell suspension was prepared by adding 492 μL of cell preservative(CELPRESOL™) to an 8 μL volume of washed, packed RBCs. An aliquot (30μL) of the cell suspension and an aliquot (30 μL) of SYPH3 positive ornegative sera were then added to a Coombs card chamber and incubated at37° C. for 30 minutes before centrifugation at automatic setting of amicrocentrifuge (DIAMED™).

A photograph of the card following centrifugation is presented as FIG. 3of the drawings pages. Serology scores were recorded as negative or apositive (+1 to +3) reaction. The serology scores are recorded in Table2. The serological performance of the kodecytes prepared according tothe method of the present invention was indistinguishable from theperformance of the kodecytes prepared by the known methods (Bovin et al(2009); Henry et al (2010)).

TABLE 1 Concentration (μg/mL) Construct Sera 200 100 50 25 0 FSL-SYPH3POS E47 +3 +3 +2 +2 0 FSL-RFG(MAL)-SYPH3 POS E47 +3 +3 +2 +1 0 FSL-SYPH3NEG E216 0 0 0 0 0 FSL-RFG(MAL)-SYPH3 NEG E216 0 0 0 0 0

The experiment was repeated including the construct designatedFSL-RFG(Mal) to confirm the observed serology scores were notattributable to cross-reactivity with this construct. The serologyscores (with no discrimination between positive serology scores) arerecorded in Table 2.

Samples (4 μL) of the reagents and constructs used were applied at aconcentration of 2 mg/mL in water to a thin layer chromatography (TLC)plate (ALUMGRA™ SIL G/UV silica 60) and eluted with 2:6:2 (v/v/v)chloroform/methanol/water containing 0.5% (v/v) pyridine. (Theconcentration of the construct designated FSL-RFG(Mal)-SYPH3 was basedon the expected concentration of the construct excluding unreacted SYPH3peptide.) The eluted TLC plate was visualised by spraying with asolution of anisaldehyde in acetic acid and heating to a temperature of200° C. A photograph of the visualised plate is presented as FIG. 4 ofthe drawings pages. The chromatographic performance of the reagents andconstructs was consistent with the construct designated FSL-SYPH3 andthe construct designated FSL-RFG(Mal)-SYPH3 being identical.

Consistent with the provision of a General Example it will beappreciated that variations and modifications may be made to the stepsof the method illustrated with reference to the Specific Example withoutdeparting from the scope of the invention. The advantages discussed inthe description may be provided in the alternative or in combination inthese other embodiments of the invention. Where known equivalents existto specific features, such equivalents are incorporated as ifspecifically referred to in this specification. In particular, it willbe understood that for a non-specific interaction, such as theinteraction between the lipid (L) moiety of the constructs (F-S-L) and asurface such as a membrane, regioisomers and stereoisomers of naturallyoccurring lipids can be functionally equivalent. For example, it iscontemplated that diacylglycerol 2-phosphate could be substituted forphosphatidate (diacylglycerol 3-phosphate) and that the absoluteconfiguration of phosphatidate could be either R or S.

REFERENCES

-   Anon (2007a) BACHEM Technical Note.-   Anon (2007b) Reversal of Inadvertent Oxidation of Cys-containing    Peptides BACHEM Technical note.-   Anon (2011) Sigma Technical Bulletins D130 and MBK1.-   Bednar (1990) Reactivity and pH dependence of thiol conjugation to    N-ethylmaleimide: detection of a conformational change in chalcone    isomerase Biochemistry, 29(15), 3684-90.-   Blake et al (2011) FSL Constructs: A simple method for modifying    cell/virion surfaces with a range of biological markers without    affecting their viability J. Vis. Exp. doi: 10.3791/3289.-   Bovin et al (2009) Functional lipid constructs International patent    application no. PCT/NZ2008/000266 (publ. no. WO 2009/048343).-   Ellman (1959) Tissue sulfhydryl group Arch. Biochem. Biophys., 82,    70-77.-   Gorin et al (1966) Kinetics of the reaction of N-ethylmaleimide with    cysteine and some congeners Arch. Biochem. Biophys., 115, 593-   Hayes et al (Eds.) (2009) Biobased Surfactants and    Detergents—Synthesis, Properties, and Applications AOCS Press.-   Henry et al (2010) Assays for serological detection of syphilis    International patent application no. PCT/NZ2010/000111 (publ. no. WO    2010/143983).-   Hermanson (Ed.)(2008) Bioconjugate Techniques Academic    Press-Elsevier-   Shafer et al (2000) Reaction of Tris(2-carboxyethyl)phosphine (TCEP)    with Maleimide and α-Haloacyl Groups: Anomalous Elution of TCEP by    Gel Filtration Analytical Biochemistry, 282, 161-4.-   Smyth et al (1960) Reactions of N-ethylmaleimide J. Am. Chem. Soc.,    82, 4600.-   Smyth et al (1964) Reaction of N-ethylmaleimide with peptides and    amino acids Biochem. J., 91, 589.-   Tyagarajan et al (2003) Thiol-reactive dyes for fluorescence    labeling of proteomic samples Electrophoresis, 24(14), 2348-58.-   Weinberg et al (2009) Peptide-lipid constructs and their use in    diagnostic and therapeutic applications International application    no. PCT/NZ2008/000239 (publ. no. WO 2009/035347).

TABLE 2 Concentration (μg/mL) Construct Sera 200 100 50 25 0 FSL-SYPH3POS E47 + + + + FSL-RFG(MAL)-SYPH3 POS E47 + + + + Nil POS E47 −FSL-SYPH3 POS E35 + − FSL-RFG(MAL)-SYPH3 POS E35 + − FSL-RFG(MAL) POSE35 − − NIL POS E35 − FSL-SYPH3 POS E36 + + FSL-RFG(MAL)-SYPH3 POSE36 + + FSL-RFG(MAL) POS E36 − − NIL POS E36 − FSL-SYPH3 POS E19FSL-RFG(MAL)-SYPH3 POS E19 + FSL-RFG(MAL) POS E19 − NIL POS E19 −FSL-SYPH3 NEG E216 − − − − FSL-RFG(MAL)-SYPH3 NEG E216 − − − − Nil NEGE216 − FSL-SYPH3 NEG E206 − − FSL-RFG(MAL)-SYPH3 NEG E206 − −FSL-RFG(MAL) NEG E206 − − Nil NEG E206 − FSL-SYPH3 NEG E226 − −FSL-RFG(MAL)-SYPH3 NEG E226 − − FSL-RFG(MAL) NEG E226 − − Nil NEG E226 −FSL-SYPH3 NEG E262 FSL-RFG(MAL)-SYPH3 NEG E262 − FSL-RFG(MAL) NEG E262 −Nil NEG E262 − FSL-SYPH3 NEG E258 FSL-RFG(MAL)-SYPH3 NEG E258 −FSL-RFG(MAL) NEG E258 − Nil NEG E258 −

INDUSTRIAL APPLICABILITY

A facile method of localising sulfhydryl containing molecules tosurfaces is disclosed. The method has particular application inbiological laboratories where access to equipment for chemicalpurification is restricted or unavailable.

1-46. (canceled)
 47. A method of localising a sulfhydryl (—SH) groupcontaining biomolecule to a cell membrane comprising the steps: a.Mixing in a volatile reaction buffer a molar excess of the sulfhydryl(—SH) group containing biomolecule with a lipid conjugated maleimide ofthe structure F-S-L to provide a reaction mix; b. Incubating thereaction mix for a time and at a temperature sufficient to allowsubstantially all the lipid conjugated maleimide to have reacted; c.Drying the reaction mix to remove the reaction buffer and provide areaction product; and d. Contacting an aqueous solution of the reactionproduct with the cell membrane, where: F is

S is selected from the group consisting of:

L is an amide linked diacyl- or dialkyl-glycerophospholipid, m is theinteger 1, 2 or 3, M is a monovalent cation or substituent, n is theinteger 1, 2 or 3, p is the integer 1, 2 or 3, q is the integer 3, 4 or5, and the median value of r is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or16.
 48. The method of claim 47 where the sulfhydryl (—SH) group is acysteine residue (Cys).
 49. The method of claim 48 where the sulfhydryl(—SH) group containing biomolecule is a peptide.
 50. The method of claim49 where the sulfhydryl (—SH) group containing biomolecule is a peptidewith a cysteine residue (Cys) at or proximal to the C-terminus of thepeptide.
 51. The method of claim 50 where the sulfhydryl (—SH) groupcontaining biomolecule is a peptide with a cysteine residue (Cys) at theC-terminus.
 52. The method of claim 51 where the surface is the cellmembrane of a red blood cell.
 53. The method of claim 47 where theincubating is for a time greater than 8 hours and at room temperature(circa 25° C.).
 54. The method of claim 47 where the drying is byfreeze-drying.
 55. The method of claim 47 where the aqueous solution ofthe reaction product is an aqueous saline solution of the reactionproduct.
 56. The method of claim 47 where the volatile reaction bufferis 0.1M 4-methylmorpholine formate (4-MMF) in 30% isopropanol (IPA) at apH in the range 6.5 to 6.7.
 57. The method of claim 47 where m is theinteger
 2. 58. The method of claim 47 where n is the integer
 2. 59. Themethod of claim 47 where p is the integer
 2. 60. The method of claim 47where q is the integer
 4. 61. The method of claim 47 where each of m, nand p are the integer 2 and q is the integer
 4. 62. The method of claim47 where the median value of r is
 6. 63. The method of claim 47 where Lis an amide linked diacylglycerophospholipid.
 64. The method of claim 63where L is an amide linked phosphatidylethanolamine.
 65. The method ofclaim 64 where L is 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine(DOPE).
 66. The method of claim 47 where S is

and M is H or CH₃.